Table 1
Primers used in quantitative RT-PCR.
| Gene | GeneBank Accession | 5’ Primer | 3’ Primer | Length |
|---|---|---|---|---|
| ARNTL2 | NM_020183 | GCTAGAGGCTACCAGGCAAAACC | GGTCCACTGGATGTCACTGAAGTC | 193 |
| NPAS2 | NM_002518 | CTTCCCTGCCTCCCAACCATC | GGTCCCTGGCTGTTGTGAGTAG | 151 |
| DEC1 | NM_003670.2 | TCAGCAGCAGCAGAAAATCATTGC | GTGGGTGACAAGCTGCGAAGAC | 187 |
| DEC2 | NM_030762 | TGCTTTACAGAATGGGGAGCGATC | CCCTGGGTGTCCAGCTCTCAAAC | 134 |
| CRY1 | NM_004075 | TCTGGCATCAGTACCTTCTAATCC | CTGTGTGTCCTCTTCCTGACTAG | 226 |
| CRY2 | NM_021117 | GGTGAAGAACTCAGCAAACGG | ACACACATGCTCGCTCTATCTC | 189 |
| DBP | NM_001352 | CTTAAGCCCCAGCCAATCATGAAG | CCGCCCGCACCGATATCTG | 160 |
| PER1 | NM_002616 | CTCCAATCAGGACGCACTTTC | GCTGCCAAAGTATTTGCTTGTG | 211 |
| PER2 | NM_022817 | TGTAGGGGCGGACTGCAAAC | TGCTGGTATGACTTGTGTCACTAC | 251 |
| PER3 | NM_016831 | TGAAGAATCCATCCCATCCTACTG | TATACTGCTGTCGCTGCTTCC | 218 |
| NR1D1 | NM_021724 | CTTGGCTGCCCAGCGTCATAAC | CCAGATCTCCTGCACCGTTCG | 274 |
| RORA | NM_134262.2 | CCAGCCCCGACGTCTTCAAAT | GCCATGAGCGATCTGCTGACA | 150 |
| JUNB | NM_002229.2 | CCACTGGGGTCCAGGGAGCA | GGACTGGGCGCAGGGTAGGA | 99 |
| IL-1β | NM_000576 | TGGCAATGAGGATGACTTGT | GGAAAGAAGGTGCTCAGGTC | 237 |
| RPLP0 | NM_001002 | GGCGACCTGGAAGTCCAACT | CCATCAGCACCACAGCCTTC | 149 |
Figure 1
TNF induces nuclear ARNTL2 expression. Human primary fibroblasts were stimulated with TNF (10 ng/ml) or PBS for 24 h. ARNTL2 is labeled red and nuclei of the cells are visualized with DAPI (blue).
Figure 2
NF-κB signaling mediates TNF induced ARNTL2 and NPAS2 expression. NF-κB signaling was blocked in human synovial fibroblasts with 1 μM IMD-0354 (abbreviated IMD) and stimulated with TNF (10 ng/ml) or PBS for 16 h. ARNTL2 and NPAS2 expressions were measured with real time PCR. Values represent means ± SEM of three different experiments performed in duplicate. *p < 0.05, **p < 0.01.
Figure 3
ARNTL2/NPAS2 dimer is potent activator of E-box element. HEK293 cells were transfected with empty vector or vector containing DEC1, DEC2, ARNTL, ARNTL2 or NPAS2 in various combinations as shown. E-box and PER3 promoter activities were analyzed using luciferase assay 48 h after the transfection. Values represent means ± SEM of three different experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
ARNTL2/NPAS2 dimer is weak inducer of PER3 and DBP in comparison with ARNTL/NPAS2. HEK293 cells were transfected with empty vector or vector containing DEC1, DEC2, ARNTL, ARNTL2 or NPAS2 in various combinations as shown for 48 h. Expression of the clock components were analyzed by quantitative PCR. Values represent means ± SEM of four different experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
DEC2 is not able to inhibit DEC1. HEK293 cells were transfected with empty vector or vector containing DEC1, DEC2, ARNTL, ARNTL2 or NPAS2 in various combinations as shown 48 h. Expression of the clock components were analyzed by quantitative PCR. Values represent means ± SEM of four different experiments performed in triplicate. *p < 0.05, ***p < 0.001.